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Predator Odor-induced Freezing Test for Mice
捕食者气味诱导的小鼠僵住试验   

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Abstract

The innate fear response is an emotional response that does not require any previously acquired conditioning. One of the standard methods to analyze the innate fear response is a 2,4,5-trimethylthiazoline (TMT)-induced freezing test. TMT is an odor originally isolated from anal secretion of the red fox. Acute TMT exposure has been shown to induce robust freezing behavior in rats and mice (Wallace and Rosen, 2000; Galliot et al., 2012). Here, I show how to expose mice to TMT and how to analyze their freezing behavior.

Keywords: Fear(恐惧), Freezing(僵住), Innate(先天的), Predator(捕食者), TMT(TMT), Odor(气味)

Background

To escape detection by predators, many mammalian species, including rodents, have developed innate fear responses triggered by odor stimuli that indicate the presence of predators (Takahashi et al., 2005). The predator’s odorous substance, such as excretion and fur particles, triggers anxiety in the rodent without direct contact and induces avoidance or freezing behavior, depending on the circumstances. For example, if a mouse is not able to run away from the source of the odor (e.g., confined to a small box), the mouse freezes. If the mouse can run away, they will avoid the source of the odor rather than freeze (Hacquemand et al., 2010; Johnston et al., 2012). TMT (2,4,5-trimethylthiazoline), a component of fox feces, is the most used synthesizable reagent for inducing innate fear in rodents (Vernet-Maury et al., 1984). Wallace et al. found that innate fear responses of rats can be quantified by measuring the freezing duration when the animals are exposed to TMT in a small confined space. They also found that innate fear responses to TMT do not induce conditioned learning. This finding indicates that different neural pathways are activated during TMT exposure from those activated during conventional footshock-induced fear responses. Lesion studies have shown that the regions associated with the innate fear responses include the medial/central nucleus of the amygdala and the bed nucleus of the stria terminalis (BNST) (Fendt et al., 2003; Müller and Fendt, 2006). Here, I present conventional methods for measuring TMT-induced fear responses in the mouse.

Materials and Reagents

  1. Kimwipe (1.5 x 2 cm)
  2. Plastic bag
  3. Gloves
  4. Disposable circular test chamber with a transparent lid (13 cm in diameter, 10 cm in height)
    Note: Transparent lid is required for video tracking. I purchased the opaque chambers (B-313, Tokyo Garasu Kikai) and modified their lids to be transparent. However totally transparent chamber could be utilized alternately.
  5. C57BL/6J from Charles River Laboratories, 14 weeks old
  6. 2,5-Dihydro-2,4,5-trimethylthiazoline (TMT) (Contech, catalog number: 13267 )
    Note: Contech no longer manufactures TMT. Obtain from other vendors (e.g., ChemSpider, catalog number: 231591 ).
  7. 80% ethanol
  8. 5% ammonium hydroxide

Equipment

  1. Charge-coupled device (CCD) camera (Logicool, model: HD WEBCAM C270 )
  2. Fume hood (Safety cabinet)
  3. Walls (Cardboard, 30 x 35 cm)
  4. Pipetman pipette (P10, Gilson)

Software

  1. ImageFZ (ImageJ plugin; Shoji et al., 2014; http://www.mouse-phenotype.org/)
    Note: Although I had used ImageFZ in the work, this software supports for only old OS (Windows XP/7 and Mac OS 9). For the compatibility with later Windows OS, a standalone software, FreezeAnalyzerForAVI (http://www.yuzaki-lab.org/publication/software) can be used as an alternative (compatible with Windows XP/7/8.1/10).

Procedure

Notes:

  1. Although TMT is not a deleterious substance, it can cause irritations at high concentrations so it should be treated in a fume hood.
  2. TMT is colorless but has a distinct, strong smell. Thus, it is not easy to perform the experiments with the experimenter blinded to the exposure condition. However, if the fume hood has a minimal opening and there is no leakage of TMT, performing this experiment in a blinded manner may be possible.
  3. Sex-and age-matched littermate controls should be used (sex, age, genetic background, and parental nurturance could affect animal behavior).
  4. The experimenter should opt for either single- or group-housing, since the number of cage mates might affect the results. I kept 2-4 mice per cage (136 x 208 x 115 mm).
  5. Avoid cage changing 24 h before the all the procedures of the experiment.
  6. Genotype/treatment should be blinded to avoid experimenter bias.
  1. Handle all animals twice for 5 min each in a fume hood the day before the TMT exposure to habituate them to the experimenter and the fume hood (Video 1).

    Video 1. An example of mice handling in the fume hood

  2. Move animal housing cages in the experimental room from the breeding area at least 30 min before start of the experiment.
    Note: If possible, experimental animal cages should be placed in a next room to experimental room to minimize an effect of leaking TMT during the experiment.
  3. Stick a piece of Kimwipe (1.5 x 2 cm) to the top edge of the wall of a disposable circular test chamber (Figure 1A).
  4. Place the test chamber in the fume hood and locate the CCD camera in a suitable position so that the test chamber is kept within its visual field (Figure 1B).


    Figure 1. Equipment for a TMT-induced freezing test. A. Disposable circular test chamber and lid; B. The placement of equipment. The chamber is placed at the center of the walled area and illuminated at 500 lux in my equipment.

  5. Place each mouse gently into the test chamber, put on a transparent lid with vents, and close the door with a blinder to hide the experimenter from the mouse. Start video capture. This ‘no-odor’ interval lasts for 5 min, in order to record a baseline level of freezing.
  6. Open the door and apply the neat 5 μl TMT to the Kimwipe with a Pipetman pipette. Close the door and continue observing for 12 min to monitor TMT-induced freezing.
    Note: Pay attention to avoid potential leakage of TMT from the fume hood to the experimental room. If TMT is spilled out, wipe out as soon as possible with 80% ethanol or 5% ammonium hydroxide.
  7. Remove the mouse from the test chamber and place it into the temporary holding chamber next to the experiment room, to prevent untested mice from being disturbed by TMT on the surface of the analyzed mouse.
  8. Replace the test chamber with a new one and seal off the used test chamber tightly in a plastic bag and place it at a separate room (Each test chamber is used only once).
  9. Renew gloves and repeat from step 5.
  10. When all experimental procedures are completed, wipe fume hood with 80% ethanol or 5% ammonium hydroxide and discard all experimental apparatus such as cardboard walls and test chambers.

Data analysis

Freezing behavior is defined as immobility with the exception of breathing and percentages of the summed freezing durations in each bin were calculated. The duration of freezing is considered to reflect a degree of fear. Longer freezing is interpreted as larger fear. Measure freezing duration of each animal by use of an automated freeze analyzing software (e.g., ImageFZ or FreezeAnalyzerForAVI), or manually in a blinded fashion. A video-rate of 2 frames/sec is sufficient for the automated analysis. A representative result of wild-type mice is shown in Video 2 and Figure 2. Two-way repeated-measures ANOVA (Genotype/treatment x 1-min time bin) was used to determine statistical difference (Wallace et al., 2000).

Video 2. An example of TMT-induced freezing. The mouse showed freezing behavior after the administration of TMT.


Figure 2. Representative result of wild-type mice in a TMT-induced freezing test. Each dot represents the percentage of average freezing duration of six female wild-types (C57BL/6J from Charles River Laboratories, 14 weeks old). Error bars show SEM. The mice were exposed to TMT for 5 min after the start of acclimation.

Acknowledgments

This protocol was modified from Galliot et al. (2012). I thank Dr. Michisuke Yuzaki for his continuous support. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H06461 to MY).

References

  1. Galliot, E., Laurent, L., Hacquemand, R., Pourie, G. and Millot, J. L. (2012). Fear-like behavioral responses in mice in different odorant environments: Trigeminal versus olfactory mediation under low doses. Behav Processes 90(2): 161-166.
  2. Fendt, M., Endres, T. and Apfelbach, R. (2003). Temporary inactivation of the bed nucleus of the stria terminalis but not of the amygdala blocks freezing induced by trimethylthiazoline, a component of fox feces. J Neurosci 23(1): 23-28.
  3. Johnston, R., Müller-Schwarze, D. and Sorenson, P. (1999). Advances in chemical signals in vertebrates. Springer.
  4. Müller, M. and Fendt, M. (2006). Temporary inactivation of the medial and basolateral amygdala differentially affects TMT-induced fear behavior in rats. Behav Brain Res 167(1): 57-62.
  5. Hacquemand, R., Jacquot, L. and Brand, G. (2010). Comparative fear-related behaviors to predator odors (TMT and natural fox feces) before and after Intranasal ZnSO4 treatment in mice. Front Behav Neurosci 4: 188.
  6. Shoji, H., Takao, K., Hattori, S. and Miyakawa, T. (2014). Contextual and cued fear conditioning test using a video analyzing system in mice. J Vis Exp (85).
  7. Takahashi, L. K., Nakashima, B. R., Hong, H. and Watanabe, K. (2005). The smell of danger: a behavioral and neural analysis of predator odor-induced fear. Neurosci Biobehav Rev 29(8): 1157-1167.
  8. Vernet-Maury, E., Polak, E. H. and Demael, A. (1984). Structure-activity relationship of stress-inducing odorants in the rat. J Chem Ecol 10(7): 1007-1018.
  9. Wallace, K. J. and Rosen, J. B. (2000). Predator odor as an unconditioned fear stimulus in rats: elicitation of freezing by trimethylthiazoline, a component of fox feces. Behav Neurosci 114(5): 912-922.

简介

天生的恐惧反应是一种情绪反应,不需要任何以前获得的条件。 分析天生恐惧反应的标准方法之一是2,4,5-三甲基噻唑啉(TMT)诱导的冷冻试验。 TMT是最初与红狐狸分泌物分离的气味。 已经显示急性TMT暴露在大鼠和小鼠中诱导强烈的冷冻行为(Wallace和Rosen,2000; Galliot等,2012)。 在这里,我将展示如何将小鼠暴露于TMT以及如何分析其冻结行为。
【背景】为了逃避捕食者的检测,许多哺乳动物物种(包括啮齿动物)已经发现了由气味刺激引发的天生恐惧反应,表明捕食者的存在(Takahashi等,2005)。捕食者的恶臭物质,例如排泄物和毛皮颗粒,会引起啮齿动物的焦虑,而不会直接接触,并根据情况引起避免或冷冻行为。例如,如果鼠标不能远离气味源(例如,限于小盒子),则鼠标将冻结。如果鼠标可以逃跑,他们将避免气味的来源而不是冻结(Hacquemand等人,2010; Johnston等人,2012)。 TMT(2,4,5-三甲基噻唑啉)是狐狸粪便的成分,是诱导啮齿动物天生恐惧的最常用的合成试剂(Vernet-Maury等,1984)。 Wallace等人发现大鼠的天生恐惧反应可以通过测量动物在小密闭空间中暴露于TMT的冻结持续时间来量化。他们还发现对TMT的天生恐惧反应不会诱导条件学习。这一发现表明,在TMT暴露期间,不同的神经通路在常规脚震引发的恐惧反应中被激活。病变研究表明,与天生恐惧反应相关的区域包括杏仁核的内侧/中心核和纹状体的核心(BNST)(Fendt et al。,2003;Müllerand Fendt,2006)。在这里,我介绍了用于在小鼠中测量TMT诱导的恐惧反应的常规方法。

关键字:恐惧, 僵住, 先天的, 捕食者, TMT, 气味

材料和试剂

  1. Kimwipe(1.5 x 2厘米)
  2. 塑料袋
  3. 手套
  4. 一次性圆形测试室,带有透明盖(直径13厘米,高10厘米) 注意:视频跟踪需要透明盖。我购买了不透明的房间(B-313,东京加拉苏Kikai),并修改了盖子是透明的。然而,完全透明的室可以交替使用。
  5. 来自Charles River实验室的C57BL / 6J,14周龄
  6. 2,5-二氢-2,4,5-三甲基噻唑啉(TMT)(Contech,目录号:13267)
    注意:Contech不再生产TMT。从其他厂商获取(例如ChemSpider,目录号:231591)。
  7. 80%乙醇
  8. 5%氢氧化铵

设备

  1. 电荷耦合器件(CCD)相机(Logicool,型号:HD WEBCAM C270)
  2. 通风柜(安全柜)
  3. 墙(纸板,30 x 35厘米)
  4. 皮皮特移液器(P10,Gilson)

软件

  1. ImageFZ(ImageJ插件; Shoji等人),2014; http://www.mouse-phenotype.org/
    注意:虽然我在工作中使用过ImageFZ,但该软件仅支持旧的操作系统(Windows XP / 7和Mac OS 9)。为了与以后的Windows操作系统兼容,独立软件FreezeAnalyzerForAVI( http://www.yuzaki-lab.org/publication/software )可以作为替代(与Windows XP / 7 / 8.1 / 10兼容)

程序

注意:

  1. 尽管TMT不是有害物质,但它可能导致高浓度的刺激,因此应在通风橱中进行处理。
  2. TMT是无色的,但具有明显的强烈的气味。因此,对于暴露条件不知情的实验者进行实验并不容易。然而,如果通风橱具有最小的开度并且没有TMT的泄漏,则可能以盲目的方式执行该实验。
  3. 应该使用性别和年龄匹配的同窝出生控制(性别,年龄,遗传背景和父母抚养可能影响动物行为)。
  4. 实验者应该选择单一或群体住房,因为笼子的数量可能会影响结果。我每笼保持2-4只小鼠(136 x 208 x 115毫米)。
  5. 在实验的所有程序之前24小时避免笼子更换。
  6. 基因型/治疗应该是盲目的,以避免实验者的偏见。
  1. 在TMT暴露之前的一天,在通风橱中处理所有动物两次,每次5分钟,以便将它们习惯于实验者和通风橱(视频1)。

    Video 1. An example of mice handling in the fume hood

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  2. 实验开始前至少30分钟,从实验室移动实验室的动物饲养笼。
    注意:如果可能,实验动物笼应放置在实验室的隔壁房间,以尽量减少实验期间TMT渗漏的影响。
  3. 将一块Kimwipe(1.5 x 2厘米)粘贴到一次性圆形测试室墙壁的顶部边缘(图1A)。
  4. 将测试室放置在通风橱中,并将CCD相机放置在合适的位置,以便测试室保持在其视野内(图1B)。


    图1. TMT诱导冷冻试验的设备。 A.一次性圆形试验箱和盖子; B.设备的放置。该房间被放置在围墙区域的中心,并在我的设备上以500勒克司灯照亮。

  5. 将每只老鼠轻轻地放入测试室,放在带通风口的透明盖子上,并用遮光罩关闭门,将实验者从鼠标中隐藏起来。开始视频捕获。这种"无气味"间隔持续5分钟,以记录基线的冻结水平。
  6. 打开门,使用Pipetman移液器将整齐的5μlTMT应用于Kimwipe。关上门,继续观察12分钟,监测TMT引起的冻结。
    注意:注意避免TMT从通风橱到实验室的潜在泄漏。如果TMT溢出,应尽快用80%乙醇或5%氢氧化铵清除。
  7. 从测试室中取出鼠标,将其放入实验室旁边的临时储存室,以防止未经测试的小鼠被分析小鼠表面的TMT干扰。
  8. 更换新的试验箱,将密封在塑料袋中的试验箱密封并放在一个独立的房间(每个试验室仅使用一次)。
  9. 续订手套,重复步骤5.
  10. 当所有实验程序完成后,用80%乙醇或5%氢氧化铵擦拭通风橱,并丢弃所有实验设备,如纸板墙和测试室。

数据分析

冻结行为被定义为除呼吸以外的不动性,并且计算每个箱中总和的冻结持续时间的百分比。冷冻时间被认为反映了一定程度的恐惧。更长的冻结被解释为更大的恐惧。通过使用自动冻结分析软件(例如,例如,ImageFZ或FreezeAnalyzerForAVI)测量每只动物的冻结持续时间,或以盲人的方式手动测量。 2帧/秒的视频速率足以进行自动分析。野生型小鼠的代表性结果显示在视频2和图2中。使用双向重复测量方差分析(基因型/处理×1分钟时间仓)来确定统计学差异(Wallace等人, / em>,2000)

Video 2. An example of TMT-induced freezing. The mouse showed freezing behavior after the administration of TMT.

To play the video, you need to install a newer version of Adobe Flash Player.

Get Adobe Flash Player



图2.野生型小鼠在TMT诱导的冷冻试验中的代表性结果。 每个点代表六个雌性野生型(C57BL / 6J来自Charles River Laboratories,14周龄)的平均冻结持续时间的百分比。错误栏显示SEM。开始适应后,将小鼠暴露于TMT 5分钟。

致谢

该协议由Galliot等人修改(2012)。我感谢Michisuke Yuzaki博士的一贯支持。这项工作得到日本教育,文化,体育,科技部(MEXT)(16H06461给MY)的支持。

参考

  1. Galliot,E.,Laurent,L.,Hacquemand,R.,Pourie,G.and Millot,JL(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm .nih.gov / pubmed / 22248569"target ="_ blank">在不同气味环境中的小鼠中的恐惧样行为反应:低剂量下的三叉神经与嗅觉调解。 2):161-166。
  2. Fendt,M.,Endres,T.and Apfelbach,R。(2003)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12514197"目标="_ blank">临时灭活纹状体的床核,而不是甲基噻唑啉引起的杏仁体块冻结,这是狐狸粪便的一个组成部分。 Neurosci 23(1) :23-28。
  3. Johnston,R.,Müller-Schwarze,D.and Sorenson,P。(1999)。脊椎动物化学信号的进展 Springer 。
  4. Müller,M。和Fendt,M。(2006)。内侧和基底外侧杏仁核的暂时失活差异影响大鼠TMT诱导的恐惧行为。 Behav Brain Res 167(1):57-62。
  5. Hacquemand,R.,Jacquot,L。和Brand,G。(2010)。< a class ="ke-insertfile"href ="https://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3013532 /"target ="_ blank">在小鼠鼻内ZnSO 4治疗之前和之后与捕食者气味(TMT和天然狐狸粪便)的比较恐惧相关行为。 Front Behav Neurosci 4:188.
  6. Shoji,H.,Takao,K.,Hattori,S.和Miyakawa,T。(2014)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov / pubmed / 24637495"target ="_ blank">使用小鼠中的视频分析系统的语境和提示的恐惧条件测试 J Vis Exp (85)。
  7. Takahashi,LK,Nakashima,BR,Hong,H.and Watanabe,K。(2005)。危险的气味:捕食者气味引起的恐惧的行为和神经分析。 Neurosci Biobehav Rev 29(8):1157-1167。 br />
  8. Vernet-Maury,E.,Polak,EH和Demael,A.(1984)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/24318845 "target ="_ blank">大鼠中应激诱导气味剂的结构 - 活性关系 Chem Chem。10(7):1007-1018。
  9. Wallace,KJ和Rosen,JB(2000)。  捕食者气味作为大鼠中无条件的恐惧刺激:通过三甲基噻唑啉诱发冻结,这是狐狸粪便的一个组成部分。 Behav Neurosci 114(5):912-922。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Otsuka, S. (2017). Predator Odor-induced Freezing Test for Mice. Bio-protocol 7(17): e2534. DOI: 10.21769/BioProtoc.2534.
  2. Fendt, M., Endres, T. and Apfelbach, R. (2003). Temporary inactivation of the bed nucleus of the stria terminalis but not of the amygdala blocks freezing induced by trimethylthiazoline, a component of fox feces. J Neurosci 23(1): 23-28.
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